The present application relates to nucleic acid constructs which can be used in genetic manipulation and in particular in the prophylaxis or therapy of diseases (termed gene therapy in that which follows). In gene therapy, genes which are to be expressed in an organism are introduced into the organism. The regulation of the expression of these genes is of significance for the prophylactic or therapeutic effect of the gene therapy.
Regulators of the expression of a gene are described in Patent Applications PCT/GB95/02000, PCT/EP95/03370, PCT/EP95/03371, PCT/EP95/03368 and PCT/EP95/03339. These regulators comprise an activator sequence whose function is, for example, the cell-specific or virus-specific activation of basal transcription. The DNA sequence of this activator sequence is linked by its 3xe2x80x2 end to the 5xe2x80x2 end of a promoter module. The structural gene is in turn linked by its 5xe2x80x2 end to the 3xe2x80x2 end of the promoter module.
The promoter module is composed of nucleic acid sequences for binding the transcription factors of the CDF and CHF families or of the E2F and CHF families. In the G0 and G1 phases of the cell cycle, this binding leads to inhibition of the upstream activator sequence and consequently to inhibition or transcription of the structural gene which is located downstream (i.e. in the direction of transcription).
In the G0 and G1 phases of cell division, the DNA which is contained in the cell is in the diploid state. In the G0 phase, the cell is at rest, while in the G1 phase its cell cycle progression is inhibited. The G1 phase is followed by the S phase, in which DNA synthesis takes place and in which the genome is replicated. There then follows the G2 phase, in which the cell is in the tetraploid state. The G2 phase is followed by cell division (mitosis=M phase). The daughter cells then pass into the G0 state or G1 state.
The combination of a cell-specific or virus-specific activator sequence and a promoter module which inhibits this activator sequence in the G0 and G1 phases consequently makes it possible to regulate the expression of a structural gene in a cell-specific or virus-specific and also cell cycle-specific (i.e. restricted to the S and G2 phases) manner.
The combination of an activator sequence and a promoter module is termed a chimeric promoter. While there are many possible applications for chimeric promoters in gene therapy, there are also a number of limitations arising from shortcomings. Examples of these limitations are:
a weak activator sequence which brings about too low a transcription of the structural gene,
the use of an activator sequence which cannot be inhibited by the chosen promoter module in a sufficiently cell cycle-dependent manner,
the restriction to two (for example cell-specific or virus-specific and cell cycle-specific) regulators of the transcription of the structural gene, and
inadequate intracellular transport of the transcription product of the structural gene which has been introduced into the cell.
The present invention overcomes the shortcomings of using known chimeric promoters to express foreign genes by providing the nucleic acid constructs of the present invention, which enable the regulated expression of foreign genes in host cells.
An object of the present invention is to make available nucleic acid constructs which enable the expression of foreign genes (transgenes) to be regulated in a precise manner in the host cells. The present invention therefore relates to nucleic acid constructs in which precise regulation of the transgene is achieved by at least one nucleic acid sequence exhibiting a first mutation which inhibits the proper expression of a transgene, and in which at least one further nucleic acid sequence exhibits a second mutation which abolishes the inhibition due to the mutation in the first nucleic acid sequence(s).
More particularly, the nucleic acid construct of the present invention regulates expression of a transgene in a host cell utilizing alternative constructs. When the nucleic acid sequence containing the first mutation is a transgene (b) containing a mutation which inhibits the transcription and/or the translation of said transgene or inhibits the function of the pharmacologically active compound, then the nucleic acid construct further comprises a first promoter or enhancer sequence (a), which is located upstream from the 5xe2x80x2 end of the transgene, or alternatively, when the nucleic acid sequence containing the first mutation is a first promoter or enhancer sequence (axe2x80x2), which contains a mutation which inhibits the function of the first promoter, then the nucleic acid construct further comprises a transgene (bxe2x80x2) encoding a pharmacologically active compound. In either instance, at least one nucleic acid sequence containing the second mutation abolishes the inhibition due to the first mutation.
These nucleotide sequences are under the control of identical or different promoter sequences, so that a transgene can only be expressed when all these promoter sequences are activated.
Preferably, the novel nucleic acid constructs comprise at least the following components, listed in the direction of reading, from the 5xe2x80x2 end to the 3xe2x80x2 end:
a first (I) promoter or enhancer sequence (a) which is nonspecific, cell-specific or virus-specific, or which can be activated by tetracycline or metabolically and/or cell cycle-specifically, which activates the transcription of a transgene and which can contain a mutation (axe2x80x2) which inhibits the function of the promoter,
a transgene (bxe2x80x2) which, as structural gene, encodes an active compound and can contain a mutation (b) which stops the transcription and/or the translation of this structural gene or inhibits the function of the product of the structural gene,
a second (II) promoter or enhancer sequence (c) or (cxe2x80x2) which is nonspecific, cell-specific or virus-specific, or which can be activated metabolically and/or cell cycle-specifically, which activates the basal transcription of the component (d) or (dxe2x80x2) and which can contain a mutation which inhibits the function of the promoter,
a gene for a tRNA (suppressor tRNA) or a regulatory protein (d) or (dxe2x80x2) for relieving the mutation in one or more of the promoters or in the transgene.
The first (I) promoter sequence or enhancer sequence (a) and the second (II) promoter sequence or enhancer sequence (c) can be identical or different, and at least one of the components (a) and (c) can be nonspecifically, cell-specifically or virus-specifically activatable, be activatable by tetracycline or metabolically, in particular by hypoxia, or be cell cycle-specifically activatable.
The invention also relates to a nucleic acid construct wherein the component (b) exhibits a nuclear retention signal whose cDNA is linked, at the 5xe2x80x2 end, directly or indirectly, to the 3xe2x80x2 end of the structural gene, and wherein the transcription product of the nuclear retention signal exhibits a structure for binding a nuclear export factor.
The invention also relates to a nucleic acid construct which, in addition to components (a) to (d), exhibits the following components:
a further promoter or enhancer sequence (i) which activates the basal transcription of a nuclear export factor, and
a nucleic acid which encodes a nuclear export factor (k) which binds to the transcription product of the nuclear retention signal (h) and thereby mediates transport of the transcription product of the transgene out of the cell nucleus into the cytoplasm.
Within the context of the present invention, at least one of the promoter sequences or enhancer sequences (a) and (c) can be a chimeric promoter in which the promoter module CDE-CHR or E2FBS-CHR can interact with an upstream activator sequence which is cell-specifically, virus-specifically or metabolically activatable and can thereby influence, in particular inhibit, the expression of a downstream gene.
The components (a) and (c) can also be activator-responsive promoter units. Such constructs also exhibit the following components:
at least one promoter or enhancer sequence (e) which can be activated nonspecifically, virus-specifically, metabolically, by tetracycline, or cell-specifically and/or cell cycle-specifically,
at least one activator subunit (f) which is situated downstream of the promoter or enhancer sequence (e) and whose transcription is activated by the promoter or enhancer sequence (e), and
an activator-responsive promoter (g) which is activated by the expression products of an activator subunit as described in (f) or of several identical or different activator subunits
In a further embodiment of the invention the nucleic acid constructs are nucleic acid constructs in which the promoter sequence or enhancer sequence (a) and/or (c) and/or (i) and/or the activator-responsive promoter (g) is a chimeric promoter and the activator subunit (f) is a gene for at least one transcription factor which activates the chimeric promoter of the activator-responsive promoter (g).
The invention also relates to a nucleic acid construct which contains an activator-responsive promoter (g) which is activated by two activator subunits (f, fxe2x80x2); e.g., the LexA operator (monomers or multimers) in conjunction with the SV40 promoter. The activator subunit (f) comprises the cDNA for the LexA DNA-binding protein, encoding amino acids 1-81 or 1-202, whose 3xe2x80x2 end is linked to the 5xe2x80x2 end of the cDNA for the Gal80 protein (amino acids 1-435). The second activator subunit (fxe2x80x2) comprises the cDNA of the Gal80-binding domain of the Gal4 protein, encoding amino acids 851-881, whose 3xe2x80x2 end is linked to the 5xe2x80x2 end of the cDNA of the SV40 large T antigen, encoding amino acids 126-132, whose 3xe2x80x2 end is linked to the 5xe2x80x2 end of the cDNA for the transactivating domain of HSV-1 VP16, encoding amino acids 406-488.
In another example of an activator-responsive promoter (g) which is activated by two activator subunits (f, fxe2x80x2), the above mentioned LexA operator is replaced with the Gal4-binding region (singly or arranged multiply in succession) and the gene for the LexA DNA-binding protein is replaced with the gene for the DNA-binding domain (AA1 to 147) of the Gal4 protein.
The invention also relates to a nucleic acid construct which contains, as the activator-responsive promoter (g), monomers and multimers of the binding sequence for the Gal4 binding protein, and the activator subunit (f) contains the nuclear localization signal (NLS) of SV40 (SV40 large T; amino acids 126-132; PKKKRKV, SEQ ID NO.: 1), the acid transactivating domain (TAD) from HSV-1 VP16 (amino acids 406488) and the cDNA for the cytoplasmic moiety of the CD4 glycoprotein (amino acids 397-435), and the activator subunit (f) contains the nuclear localization signal (NLS) of SV40 (SV40 large T; amino acids 126-132; PKKKRKV; SEQ ID NO. 1), the cDNA for the DNA-binding domain of the Gal4 protein (amino acids 1-147) and the cDNA for the CD4-binding sequence of the p56 Ick protein (amino acids 1-71).
In another example of an activator-responsive promoter unit in which the activator-responsive promoter (g) is the binding sequence for Gal4, the cDNA for the Gal80 protein (amino acids 1435) in the activator subunit (f) is replaced with the cDNA for the cytoplasmic moiety of the CD4 glycoprotein (amino acids 397-437; Simpson et al., Oncogene 4: 1141 (1989); Maddon et al., Cell 42: 93 (1985)) and the cDNA of the Gal80-binding domain of the Gal4 protein (encoding amino acids 851-881) in the activator subunit (f) is replaced with the cDNA for the CD4-binding sequence of the p56 Ick protein (amino acids 1-71; Shaw et al., Cell 59: 627 (1989); Turner et al., Cell 60: 755 (1990); Perlmutter et al., J. Cell. Biochem. 38: 117 (1988)).
In a further preferred embodiment, the novel nucleic acid construct can exhibit a nuclear retention signal (NRS) which is linked, downstream in the reading direction (i.e. by the 5xe2x80x2 end of its DNA), to a transgene (b), (i.e. at the 3xe2x80x2 end of the transgene).
In another preferred embodiment, the transcription product of the nuclear retention signal has a structure for binding a nuclear export factor (NEF). The cDNA for this nuclear export factor is preferably linked, by its 5xe2x80x2 end, to the 3xe2x80x2 end of another promoter sequence or enhancer sequence which can be identical to or different from the promoter sequences (a) and/or (c).
The nuclear export factor (k) is preferably a gene which is selected from the group consisting of the rev gene of retroviruses such as the HIV-1 or HIV-2 viruses, visna-maedi virus, caprine arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus or HTLV, or the gene for the hnRNP-A1 protein, or the gene for the transcription factor TFIII-A.
As a rule, the nucleic acid is DNA. The novel nucleic acid constructs are customarily employed as vectors, in particular plasmid vectors (non-viral) or viral vectors.
As a rule, the transgene is a structural gene which encodes a pharmacologically active compound which is selected from the group consisting of cytokines, interferons, growth factors, antibodies or antibody fragments, receptors for cytokines or growth factors, proteins having an antiproliferative or cytostatic effect, enzymes, angiogenesis inhibitors, thrombosis-inducing substances and coagulation inhibitors, proteins having a fibrinolytic effect, blood plasma proteins, complement-activating proteins, virus coat proteins, bacterial antigens and parasitic antigens, tumor antigens, proteins having an effect on the blood circulation, peptide hormones and ribonucleic acids, such as ribozymes and antisense RNA.
In a particular embodiment, the transgene can be a structural gene which encodes a protein which triggers controlled cell death. An example of these proteins is sphingomyelinase.
In another embodiment, the transgene (b) can be a structural gene which encodes an enzyme which cleaves a precursor of a drug to form a drug. In a particular embodiment, the transgene can be a structural gene which encodes a fusion protein which is composed of a ligand and one of the previously mentioned proteins or peptide active compounds. The ligand can, for example, be an antibody, an antibody fragment, a cytokine, a growth factor, a peptide hormone or a receptor. In a particular embodiment, the structural gene can encode a ligand-enzyme fusion protein, with the enzyme cleaving a precursor of a drug, thereby forming a drug, and the ligand binding to a cell surface, preferably on endothelial cells or tumor cells.
The promoter sequence, enhancer sequence or activator sequence can be selected from the group of gene-regulatory nucleotide sequences which activate in endothelial cells, smooth muscle cells, striated muscle cells, macrophages, lymphocytes, tumor cells, liver cells, leukemia cells and glia cells, or of promoter sequences from the HBV, HCV, HSV, HPV, EBV, HTLV or HIV viruses.
The activator sequence can furthermore be a tetracycline operator in combination with a corresponding repressor.
The invention relates to viral or non-viral vectors which contain a novel nucleic acid construct and which are locally or perorally administered to, or injected into patients. Additionally the novel nucleic acid construct can also be administered intravenously, intraarterially, into a body cavity, into an organ or subcutaneously.
The invention also relates to isolated cells or cell lines which harbor a novel nucleic acid construct and which are locally administered to, or injected into patients.
Examples of such cells are tumor cells, immune cells such as a macrophage or a lymphocyte, or endothelial cells. Cells of this nature can also be used for preparing a pharmaceutical for treating a disease, with the preparation of the pharmaceutical comprising the introduction of the nucleic acid construct into a target cell.
The novel nucleic acid constructs allow any promoters, enhancers or activator sequences to be used.
The novel mutation in or on the transgene (b) can be the replacement of the nucleic acid sequence for one or more amino acids such that, as a result of this replacement, the expressed protein is no longer capable of functioning. In this case, the component (d) is a nucleic acid sequence which encodes a tRNA which, on the one hand, binds by its anticodon to the mRNA of the mutated nucleotide sequence in the transgene (b) and, on the other hand, carries an end group which takes up the correct amino acid for relieving the mutation in the transgene (b).
However, the novel mutation in or on the transgene (b) can also be a translation stop codon in the structural gene, which codon is either not found or only rarely found in mammalian cells, such that the structural gene is not effectively translated. In this case, the component (d) is a nucleic acid sequence which, on the one hand, encodes a tRNA which possesses an anticodon which is complementary to the stop codon and thereby relieves the inhibition of the translation which is due to the translation stop codon in the structural gene (b) and, on the other hand, carries an end group which takes up the correct amino acid for relieving the mutation in the transgene (b).
In another embodiment, the mutation in or on the transgene (b) can be a mutation of the TATA box of a promoter sequence which is located upstream of the 5xe2x80x2 end of the structural gene. This mutation blocks the initiation of the transcription of the structural gene. In this case, the component (d) is a nucleic acid sequence which encodes a protein which binds to the mutated TATA box and thereby enables transcription to take place.
The present invention is further directed to a method of inhibiting cell proliferation by contacting cells with a cell proliferation inhibiting amount of the nucleic acid construct containing at least one nucleic acid sequence containing a first mutation which inhibits the proper expression of a transgene, and at least one nucleic acid sequence containing a second mutation which abolishes the inhibition due to the first mutation.
The present invention also is directed to a method of treating a subject having a disease involving excessive cell proliferation, wherein the method comprises administering to the subject a cell proliferation inhibiting amount of the nucleic acid construct of the present invention. The diseases for which the nucleic acid constructs are particularly useful is in the treatment of tumors and cardiovascular diseases involving proliferation of cells in blood vessels.
The present invention is additionally directed to a pharmaceutical composition containing a cell proliferation inhibiting amount of the nucleic acid construct in a pharmaceutically acceptable carrier.
The present invention is also directed to a method of treating a subject having any one of the following medical conditions: autoimmune disease, allergies, inflammation, organ rejection, arthritis, infectious disease or neuron disease, wherein the method comprises administering to a subject a medical condition treating amount of the nucleic acid construct of the present invention.
The nucleic acid constructs disclosed in the figures are merely examples of preferred embodiments and are not meant to limit the invention to the specific components disclosed therein.